Immunoassay employing two-step internal calibration reaction

ABSTRACT

Methods for quantitatively measuring the amount of an analyte of interest in a fluid sample, and kits useful in the methods, are disclosed. The methods comprise sandwich assays, and utilize an internal calibration reaction that closely mimics the reaction of test particles by the use of a two-step reaction.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2005/042668, which designated the United States and was filed onNov. 22, 2005, published in English, which claims the benefit of U.S.Provisional Application No. 60/630,866, filed on Nov. 23, 2004. Theentire teachings of the above applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Quantitative analysis of cells and analytes in fluid samples,particularly bodily fluid samples, often provides critical diagnosticand treatment information for physicians and patients. Quantitativeimmunoassays utilize the specificity of the antigen (Ag)-antibody (Ab)reaction to detect and quantitate the amount of an Ag or Ab in a sample.In solid phase immunoassays, one reagent (e.g., the Ag or Ab) isattached to a solid surface, facilitating separation of bound reagentsor analytes from free reagents or analytes. The solid phase is exposedto a sample containing the analyte, which binds to its Ag or Ab; theextent of this binding is quantitated to provide a measure of theanalyte concentration in the sample. Transduction of the binding eventinto a measurable signal, however, is affected by a number oflimitations, including constraints of particle movement on the solidphase, which affect the specificity and applicability of quantitativeimmunoassays

SUMMARY OF THE INVENTION

The present invention pertains to methods for quantitatively measuringthe amount of an analyte of interest in a sample. The methods employ ananalyte binding agent, which can be a member of a specific binding pairtogether with the analyte of interest. In certain embodiments, theanalyte binding agent can be, for example, an antibody; an antibodyfragment; a hapten; a drug conjugate; or a receptor. The methods alsoemploy a calibration analyte and the calibration analyte binding agent;these can also be members of a specific binding pair. In certainembodiments, the calibration analyte binding agent can be, for example,an antibody; an antibody fragment; a hapten; a drug conjugate; or areceptor. If the analyte binding agent is an antibody, the samplecapture reagent can also be an antibody, such as an antibody directedagainst the same epitope of the analyte as the antibody on the bindingparticles, or an antibody directed against a different epitope of theanalyte as the antibody on the binding particles. If the calibrationanalyte binding agent is an antibody, the calibration capture reagentcan be, for example, an antibody directed against the same epitope ofthe calibration analyte as the antibody on the binding particles, or anantibody directed against a different epitope of the calibration analyteas the antibody on the binding particles.

The methods also utilize a solid phase apparatus comprising a membranestrip comprising an application point, a sample capture zone and acalibration capture zone, wherein the sample capture zone is between thecontact region and the calibration capture zone; and a sample collectionapparatus that is not in fluid contact with the solid phase apparatus.The sample collection apparatus contains either: i) a population ofbinding particles, wherein the binding particles are coated with both ananalyte binding agent and a calibration analyte binding agent; or ii) apopulation of analyte binding particles, wherein the analyte bindingparticles are coated with an analyte binding agent, and a population ofcalibration binding particles, wherein the calibration binding particlesare coated with a calibration binding agent. The sample collectionapparatus may also contain a calibration analyte; if so, the populationof particles is located at a different place in the sample collectionapparatus from the calibration analyte. In certain embodiments, thebinding particles, or analyte binding particles and calibration bindingparticles, are evaporatively-dried, vacuum-dried or freeze-dried in thesample collection apparatus. The calibration analyte can also beevaporatively-dried, vacuum-dried or freeze-dried in the samplecollection apparatus.

To perform the assays, the sample to be assayed for the analyte ofinterest is introduced into the sample collection apparatus, and abuffer is subsequently introduced into the sample collection apparatus;alternatively, a buffer is introduced into the sample collectionapparatus, and subsequently the sample is introduced into the samplecollection apparatus. If there is no calibration analyte in the samplecollection apparatus, then the buffer contains calibration analyte. Thebuffered, mixed fluid sample contains contacted binding particles (e.g.,binding particles having calibration analyte bound thereon and/or havinganalyte of interest bound thereon; and/or binding particles havingneither calibration analyte nor analyte of interest bound thereon; oranalyte binding particles having analyte of interest bound thereon;analyte binding particles not having analyte of interest bound thereon;calibration binding particles having calibration analyte bound thereon;and calibration binding particles not having calibration analyte boundthereon).

The buffered, mixed fluid sample is applied to the application point ofthe membrane strip, and the strip is maintained under conditions whichallow fluid to transport contacted binding particles by capillary actionthrough the strip to and through the sample capture zone that has asample capture reagent immobilized thereon. The sample capture reagentcan bind to analyte of interest, and thereby can interact with bindingparticles having analyte of interest bound thereon. The strip is furthermaintained under conditions which allow the fluid in the sample totransport contacted binding particles by capillary action through thestrip to and through the calibration capture zone that has a calibrationcapture reagent immobilized thereon. The calibration capture reagent canbind to the calibration analyte, and thereby can interact with bindingparticles having calibration analyte bound thereon. The membrane stripis further maintained under conditions which allow the fluid in thesample to transport contacted binding particles not bound to the samplecapture reagent or to the calibration capture reagent by capillaryaction beyond the calibration capture zone, such as into a wicking pad.

To determine the amount of analyte of interest in the sample, the amountof binding particles in the sample capture zone and the amount ofbinding particles in the calibration capture zone are assessed, and acorrected binding particle amount is determined from the amount ofbinding particles in the sample capture zone and the amount of bindingparticles in the calibration capture zone. The amount of analyte ofinterest in the sample is directly related to the corrected bindingparticle amount. In certain embodiments, the corrected binding particleamount is determined as a ratio of the amount of binding particles inthe sample capture zone, to the amount of binding particles in thecalibration capture zone; in other embodiments, the corrected bindingparticle amount is determined as a ratio of the amount of bindingparticles in the sample capture zone, to the sum of the amount ofbinding particles in the calibration capture zone and the amount ofbinding particles in the sample capture zone.

The assays of the present invention provide an internal calibrationreaction that closely mimics the reaction of the test particles by theuse of a two-step reaction, and thereby allows more accuratedetermination of the amount of an analyte of interest in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a graphic depiction of ratios of particles detected at thesample capture zone, to those detected at the calibration capture zone,indicating the presence of compensation for temperature effects. TL=TestLine (sample capture zone); ISL=Internal Standard Line (calibrationcapture zone); R10=Ratio (TL/TL+ISL).

FIG. 2 is a graphic depiction of the effect of temperature on capture ofparticles at the sample capture zone.

FIG. 3 is a graphic depiction of the effect of temperature on capture ofparticles at the calibration capture zone.

FIG. 4 is a graphic depiction of ratios of particles detected at thesample capture zone, to those detected at the calibration capture zone,indicating the presence of compensation for temperature effects. TL=TestLine (sample capture zone); ISL=Internal Standard Line (calibrationcapture zone); R10=Ratio (TL/TL+ISL).

FIG. 5 is a graphic depiction of the effect of temperature on capture ofparticles at the sample capture zone for a variety of concentrations ofanalyte.

FIG. 6 is a graphic depiction of the effect of temperature on capture ofparticles at the calibration capture zone for a variety ofconcentrations of analyte.

FIG. 7 is a graphic representation of fluorescence at the sample capturezone and the control capture zone for an assay detecting B-typeNatriuretic protein (BNP).

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The present invention pertains to methods of quantitatively measuringthe amount of an analyte in a sample using assays, particularlyquantitative immunochromatographic assays, and kits therefor.

An “assay,” as used herein, refers to an in vitro procedure for analysisof a sample to determine the presence, absence, or quantity of one ormore analytes of interest. The assays of the inventions utilize ananalyte binding agent; the analyte of interest and the analyte bindingagent are members of a specific “binding pair,” in which a first memberof the binding pair (e.g., analyte) reacts specifically with a secondmember (e.g., the binding agent). Specific interaction between themembers of the binding pair indicates that the first member of thebinding pair preferentially binds or otherwise interacts with the secondmember of the binding pair, preferably to the exclusion of any bindingto another compound in the assay. Together, the analyte of interest andthe analyte binding agent are referred to herein as the “analytespecific binding pair” One or both members of the analyte specificbinding pair can be an antibody. For example, a first member of thebinding pair (e.g., an analyte of interest) can be an antibody, and asecond member of the binding pair (e.g., a binding agent) can beanti-immunoglobulin antibody. Alternatively, the first member of thebinding pair (e.g., the analyte) can be an antigen, and the secondmember of the binding pair (e.g., the binding agent) can be an antibody;or the first member of the binding pair (e.g., the analyte) can beantibody, and the second member of the binding pair (e.g., the bindingagent) can be an antigen.

The assays of the inventions additionally utilize a calibration analyteand a calibration analyte binding agent. The calibration analyte and thecalibration analyte binding agent are similarly members of a specificbinding pair in which the first member of the binding pair (e.g.,calibration analyte) reacts specifically with the second member (e.g.,the calibration analyte binding agent). As with regard to the analyteand the analyte binding agent, one or both members of the binding pairof the calibration analyte and the calibration analyte binding agent(together, the “calibration specific binding pair”) can be an antibody.For example, a first member of the binding pair (e.g., the calibrationanalyte) can be an antibody, and a second member of the binding pair(e.g., the calibration analyte binding agent) can be anti-immunoglobulinantibody; alternatively, the first member of the binding pair (e.g., thecalibration analyte) can be an antigen, and the second member of thebinding pair (e.g., the calibration analyte binding agent) can be anantibody.

Alternatively, in other embodiments of the assays of the invention, noneof the members of the analyte specific binding pair nor the calibrationspecific binding pair need be antibodies. For example, in oneembodiment, the first member of a binding pair can be a ligand, and thesecond member of that binding pair can be a receptor; alternatively, thefirst member of an binding pair can be a lectin, and the second memberof that binding pair can be a sugar. In still another embodiment, thefirst member of a binding pair can be a nucleic acid (e.g., DNA, RNA),and the second member of that binding pair can be a nucleic acid whichspecifically hybridizes to the first member of that binding pair.

“Specific hybridization,” as used herein, refers to the ability of afirst nucleic acid to hybridize to a second nucleic acid in a mannersuch that the first nucleic acid does not hybridize to any nucleic acidother than to the second nucleic acid (e.g., when the first nucleic acidhas a higher similarity to the second nucleic acid than to any othernucleic acid in a sample wherein the hybridization is to be performed).“Stringency conditions” for hybridization is a term of art which refersto the incubation and wash conditions, e.g., conditions of temperatureand buffer concentration, which permit hybridization of a particularnucleic acid to a second nucleic acid; the first nucleic acid may beperfectly (i.e., 100%) complementary to the second, or the first andsecond may share some degree of complementarity which is less thanperfect (e.g., 70%, 75%, 80%, 85%, 90%, 95%). For example, certain highstringency conditions can be used which distinguish perfectlycomplementary nucleic acids from those of less complementarity.

“High stringency conditions”, “moderate stringency conditions” and “lowstringency conditions” for nucleic acid hybridizations are explained onpages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols inMolecular Biology (Ausubel, F. M. et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, (1998), the entire teachings ofwhich are incorporated by reference herein). The exact conditions whichdetermine the stringency of hybridization depend not only on ionicstrength (e.g., 0.2×SSC, 0.1×SSC), temperature (e.g., room temperature,42° C., 68° C.) and the concentration of destabilizing agents such asformamide or denaturing agents such as SDS, but also on factors such asthe length of the nucleic acid sequence, base composition, percentmismatch between hybridizing sequences and the frequency of occurrenceof subsets of that sequence within other non-identical sequences. Thus,equivalent conditions can be determined by varying one or more of theseparameters while maintaining a similar degree of identity or similaritybetween the two nucleic acid molecules.

In the methods of the invention, an assay is used. In one embodiment,the assay is an “immunoassay” which utilizes antibodies as a componentof the procedure. In a preferred embodiment, the assay is a “sandwich”assay, which is a test for an analyte in which a fluid containing sampleto be assessed for the presence or absence, or quantity of analyte, iscontacted with particles coated with an analyte binding agent, such asantibodies to the analyte, and the resultant mixture is applied to amembrane and subsequently moves by capillary action through themembrane. A positive result is indicated by detection of interactionbetween analyte and analyte binding agent-coated particles in a capturezone of the membrane, the amount of analyte binding agent-coatedparticles in the capture zone being related to the amount of analyte inthe fluid sample.

In the methods of the invention, a sample is assessed for the presenceor absence, or quantity, of an analyte of interest. The term “analyte ofinterest,” as used herein, refers to a first member of an analytespecific binding pair as described above. The analyte is a molecule orcompound for which the amount will be measured. The analyte can be inthe form of a solid, such as a dry substance (e.g., a powder, aparticulate; spore; or other particle), or can be in the form of a fluid(e.g., a solid as described above that has been dissolved or suspendedin a fluid; or other liquid sample). Examples of analytes includespores; proteins, such as hormones or enzymes; glycoproteins; peptides;small molecules; polysaccharides; lectins; antibodies; antibodyfragments; nucleic acids; drugs; drug conjugates; toxins (e.g.,environmental toxins); viruses or virus particles; bacteria; otherinfectious agents or products of infectious agents; portions of a cellwall; haptens; receptors; and other compounds. In a preferredembodiment, the analyte is “immunogenic,” which indicates thatantibodies (as described below) can be raised to the analyte, or to ananalyte that is bound to a carrier (e.g., a hapten-carrier conjugate,for which antibodies can be raised to the hapten). In somerepresentative embodiments, the analyte of interest can be myoglobin;CK-MB; troponin I; PSA; digoxin; theophylline; ricin; C-reactiveprotein; or b-natriuretic peptide. In other representative embodiments,the analyte of interest can be a hormone (e.g., T-3 or T-4) or a drug ofabuse (LSD, THC, barbiturates, etc.). In still other representativeembodiments, the analyte of interest can be an infectious agent or aproduct of an infectious agent, such as Francisella tularensis (thecausative agent of tularemia); Claustridia or toxin produced thereby(botulinum toxin); Variola (smallpox) virus or other pox viruses (e.g.,cowpox, monkey pox); or a spore of a type of Bacillus, such as Bacillusanthracis (anthrax) or Bacillus globigii. The analyte of interest can bein a liquid sample; alternatively, the analyte of interest can be in adry (non-fluid) sample (e.g., a solid, such as a particulate sample,powder sample, or soil sample).

If the sample that is assessed for the presence or absence, or quantity,of the analyte of interest, is a fluid, it is a fluid that wets themembrane material; that supports a reaction between the analyte ofinterest and the analyte binding agent, such as the antibody/antigenreaction (i.e., does not interfere with antibody/antigen interaction);and that has a viscosity that is sufficiently low to allow movement ofthe fluid by capillary action. In a preferred embodiment, the fluid isan aqueous solution (such as a bodily fluid). A fluid sample can be afluid having relatively few components, for example, an aqueous solutioncontaining the analyte of interest; alternatively, a fluid sample can bea fluid having many components, such as a complex environmental sample(e.g., sewage, waste water, groundwater, or other water sample), or acomplex biological fluid (e.g., whole blood, plasma, serum, urine,cerebrospinal fluid, saliva, semen, vitreous fluid, synovial fluid, orother biological fluid). In a preferred embodiment in which the fluid isa biological fluid, the fluid is whole blood, plasma, or serum. Ifdesired, the fluid sample can be diluted; for example, if a complexbiological fluid is used as the fluid sample, it can be diluted with asolution (e.g., an aqueous solution).

If the analyte of interest is not in solution (e.g., the analyte ofinterest is in a dry or solid sample, as described above), it can beextracted, suspended, or dissolved into a fluid. For example, if theanalyte of interest is a nucleic acid, it can be extracted from cells ofinterest into a solution (e.g., an aqueous solution, such as the bufferdescribed below); in another example, if the analyte of interest is apowder or particulate material (e.g., a powder, a particulate, a soilsample, or spores), it can be suspended or dissolved into a solution(e.g., an aqueous solution, such as the buffer described below) such asby obtaining a sample of the dry material (e.g., using a swab or otherinstrument) and placing the sample of dry material into the solution.Thus, a “fluid sample” can refer not only to a liquid sample to beassessed for an analyte of interest, but also to a fluid in which asolid material sample (to be assessed for an analyte of interest) isextracted, suspended or dissolved.

The “analyte binding agent,” as used herein, refers to second member ofa binding pair as described above. The analyte binding agent is acompound that specifically binds to the analyte (the first member of thebinding pair), such as an antibody, a hapten or drug conjugate, areceptor, or another binding partner. In a preferred embodiment, theanalyte binding agent is an antibody to the analyte of interest.

In a preferred embodiment of the invention, the analyte of interest andthe calibration analyte have similar properties: for example, if theanalyte of interest is a protein, the calibration analyte is a differentprotein. In another preferred embodiment of the invention, the analytebinding agent and the calibration analyte binding agent also havesimilar properties: for example, if the analyte binding agent is anantibody, the calibration analyte binding agent is also an antibody. Inaddition, the calibration analyte is an agent that is unlikely to appearin the sample being tested for the analyte of interest (for example, ifthe sample containing the analyte of interest is a blood sample, thecalibration analyte preferably is an agent, such as a protein, that isnot typically found in a blood sample), and is an agent that will not becross-reactive with the analyte binding agent.

The “sandwich” assay of the invention utilizes a solid phase apparatus.The solid phase apparatus includes a membrane strip having anapplication point, a sample capture zone, and a calibration capturezone. The solid phase apparatus may optionally include a wicking padfollowing the calibration capture zone, and a sample pad adjacent to orcovering the application point. The membrane strip can be made of asubstance having the following characteristics: sufficient porosity toallow capillary action of fluid along its surface and through itsinterior; the ability to allow movement of coated particles (e.g.,analyte binding particles, as described below) or complexes of particlesand analyte of interest (e.g., contacted analyte binding particles, asdescribed below) by capillary action (i.e., it must not block theparticles or complexes of particles and analyte of interest); and theability to be wet by the fluid containing the analyte. Examples ofmembrane substances include: cellulose, cellulose nitrate, celluloseacetate, glass fiber, nylon, polyelectrolyte ion exchange membrane,acrylic copolymer/nylon, and polyethersulfone. In a preferredembodiment, the membrane strip is made of cellulose nitrate (e.g., acellulose nitrate membrane with a Mylar backing) or of glass fiber.

The “application point” is the position on the membrane where a fluidcan be applied. An “application pad” can also optionally be used. Forexample, if the sample containing the analyte of interest containsparticles or components that should preferentially be excluded from theimmunoassay, an application pad can be used. The application padtypically can filter out particles or components that are larger (e.g.,greater than approximately 2 to 5 microns) than the particles used inthe methods of the invention (e.g., approximately 0.3-X microns) In oneembodiment, for example, the sample containing the analyte of interestis a blood sample; to remove red blood cells, white blood cells, andother components, an application pad is used. Alternatively, forexample, if the sample is an environmental or “dirty” sample, anapplication pad is used to substantially remove larger particles. If anapplication pad is used, it rests on the membrane, immediately adjacentto or covering the application point. The application pad can be made ofan absorbent substance which can deliver a fluid sample, when applied tothe pad, to the application point on the membrane. Representativesubstances include cellulose, cellulose nitrate, cellulose acetate,nylon, polyelectrolyte ion exchange membrane, acrylic copolymer/nylon,polyethersulfone, or glass fibers. In one embodiment, the pad is aHemasep®-V pad (Pall Corporation). In another embodiment, the pad is aPall 133, Pall A/D, or glass fiber pad.

If a wicking pad is present, as described above, it can similarly bemade from such absorbent substances. In a preferred embodiment, awicking pad is used, as it allows continuation of the flow of liquid bycapillary action past the capture zones and facilitates the movement ofnonbound particles away from the capture zones.

The “sample capture zone” refers to a point on the membrane strip atwhich a “sample capture reagent” is immobilized (e.g., coated on and/orpermeated through the membrane). The sample capture reagent is ananalyte binding agent, such as those described above. The sample capturereagent need not be the same analyte binding agent as described above;however, the sample capture reagent also forms a binding pair with theanalyte of interest, in that it specifically and preferentially binds tothe analyte of interest. In a preferred embodiment, the sample capturereagent is an antibody directed against the analyte; it can be directedagainst the same epitope of the analyte as, or against a differentepitope of the analyte from, the epitope that binds to the antibodiesused as analyte binding agents coated on the particles. For example, incertain embodiments in which the analyte expresses a multitude of anepitope (e.g., anthrax, a large bacterium that expresses many of thesame antigenic epitopes on its surface), the sample capture reagent canbe an antibody directed against the same epitope of the analyte as theepitope that is targeted by the analyte binding agents on the particles.In another example, in certain embodiments in which the analyte does notexpress a multitude of an epitope (e.g., troponin I, a small moleculethat expresses a few different antigenic epitopes on its surface), thesample capture reagent can be an antibody directed against a differentepitope of the analyte from the epitope that is targeted by the analytebinding agents on the particles.

In one preferred embodiment in which the analyte of interest is anantibody to an antigen, the analyte binding agent is antigen, and thesample capture reagent is an antibody to the antibody that is theanalyte of interest. In this embodiment, there should be an excess ofthe sample capture reagent, because it will capture not only antibodythat is bound to the antigen on the binding particles, but also to“free” antibody that is not bound to any particles.

The apparatus additionally includes a “calibration capture reagent”immobilized (e.g., coated on and/or permeated through the membrane) in a“calibration capture zone.” The calibration capture reagent interactswith the calibration analyte, and does not interact with the analyte ofinterest. The calibration capture reagent is a calibration analytebinding agent, such as those described above. The calibration capturereagent need not be the same calibration analyte binding agent asdescribed above; however, the calibration capture reagent also forms abinding pair with the calibration analyte, in that it specifically andpreferentially binds to the calibration analyte. In a preferredembodiment, the calibration capture reagent is an antibody directedagainst the calibration analyte; it can be directed against the sameepitope of the calibration analyte as, or against a different epitope ofthe calibration analyte from, the epitope that binds to the antibodiesused as calibration analyte binding agents coated on the particles. Incertain embodiments in which the calibration analyte expresses amultitude of an epitope, the calibration capture reagent can be anantibody directed against the same epitope of the calibration analyte asthe epitope that is targeted by the calibration analyte binding agent onthe calibration analyte binding particles (as described below). Inanother example, in certain embodiments in which the calibration analytedoes not express a multitude of an epitope, the calibration capturereagent can be an antibody directed against a different epitope of thecalibration analyte from the epitope that is targeted by the calibrationanalyte binding agents on the calibration analyte binding particles.

The calibration capture zone is positioned such that the sample capturezone is between the application point and the calibration capture zone.In a preferred embodiment, the calibration capture zone is closelyadjacent to the sample capture zone, so that the dynamics of thecapillary action of the components of the assay are similar (e.g.,essentially the same) at both the calibration capture zone and thesample capture zone. For example, the two capture zones are sufficientlyclose together such that the speed of the liquid flow is similar overboth zones. Although they are closely adjacent, the calibration capturezone and the sample capture zone are also sufficiently spaced such thatthe particles arrested in each zone can be quantitated individually(e.g., without cross-talk). Furthermore, in a preferred embodiment, thesample capture zone is separated from the application point by a spacethat is a large distance, relative to the small distance between thesample capture zone and the calibration capture zone. Because particlecapture is a rate limiting step in the assay, the distance between theapplication point and the capture zones (where particles are captured)must be sufficient to retard the speed of the liquid flow to a rate thatis slow enough to allow capture of particles when the liquid flow movesover the sample capture zone. The optimal distances between thecomponents on the membrane strip can be determined and adjusted usingroutine experimentation.

The quantitative assay additionally uses a sample collection apparatus.The sample collection apparatus is not in fluid contact with the solidphase apparatus “Not in fluid contact,” as used herein, indicates thatfluid will not flow passively from the sample collection apparatusonto/into application point; rather, a positive action is necessary toallow fluid flow. For example, physical separation (separate location)or separation by a physical component (e.g., a membrane, a hydrophobicregion that would prevent liquid flow) can be used.

A “sample collection apparatus,” as used herein, refers to an apparatusthat can be used for collection of the fluid sample or into which acollected fluid sample can be deposited or stored. The sample collectionapparatus can be any apparatus which can contain binding particles asdescribed below (e.g., binding particles, analyte binding particles,calibration analyte binding particles), and which can be added ameasured volume of fluid sample. Representative sample collectionapparatus include a sample tube, a test tube, a vial, a pipette orpipette tip, a syringe. In a preferred embodiment, the sample collectionapparatus is a pipette or pipette tip.

In one embodiment, the sample collection apparatus contains a populationof “binding particles” which are coated with the analyte binding agentand with the calibration analyte binding agent. In another embodiment,the sample collection apparatus contains a population of “analytebinding particles,” which are coated with the analyte binding agent, anda population of “calibration analyte binding particles,” which arecoated with the calibration analyte binding agent. In either embodiment,the population of particles varies, depending on the size andcomposition of the particles, the composition of the membrane of thesolid phase apparatus, and the level of sensitivity of the assay. Thepopulation typically ranges approximately between 1×10³ and 1×10⁹,although fewer or more can be used if desired. In certain embodimentsthe amount of particles is determined as an amount of solids in thesuspension used to apply the particles for storage within the samplecollection apparatus, as described below. For example, when applying theparticles in solution for freeze- or vacuum-drying in the samplecollection apparatus, a suspension of approximately 0.05% to 0.228%solids (W/V) in 5 μl of suspension can be used. Alternatively, otheramounts can be used, including, for example, from approximately 0.01% to0.5% (W/V).

The binding particles are particles which can be coated with the analytebinding agent (the second member of the analyte binding pair) and/orwith the calibration analyte binding agent (the second member of thecalibration analyte binding pair). In a preferred embodiment, theanalyte binding particles are liposomes, colloidal gold, organic polymerlatex particles, inorganic fluorescent particles or phosphorescentparticles. In a particularly preferred embodiment, the particles arepolystyrene latex beads, and most particularly, polystyrene latex beadsthat have been prepared in the absence of surfactant, such assurfactant-free Superactive Uniform Aldehyde/Sulfate Latexes(Interfacial Dynamics Corp., Portland, Oreg.).

The size of the particles is related to porosity of the membrane (foranalytes in fluid samples) and also to the size of the analyte ofinterest (e.g., for particulate analytes): the particles must besufficiently small to be transported along the membrane by capillaryaction of fluid, and also (for solid, e.g., particulate analytes,sufficiently small for the complex of contacted binding particles, asdescribed below, to be transported along the membrane by capillaryaction). The particles can be labeled to facilitate detection. Theparticles are labeled by a means which does not significantly affect thephysical properties of the particles; for example, the particles arelabeled internally (that is, the label is included within the particle,such as within the liposome or inside the polystyrene latex bead).Representative labels include luminescent labels; chemiluminescentlabels; phosphorescent labels; fluorescent labels; phosphorescentlabels; enzyme-linked labels; chemical labels, such as electroactiveagents (e.g., ferrocyanide); and colorimetric labels, such as dyes. Inone embodiment, a fluorescent label is used. In another embodiment,phosphorescent particles are used, particularly “up-converting”phosphorescent particles, such as those described in U.S. Pat. No.5,043,265.

In a first embodiment, the collection apparatus contains particles thatare coated with an analyte binding agent that is a second member of theanalyte binding pair. As described above, the analyte binding agent(second member of the analyte binding pair) specifically andpreferentially binds to the analyte of interest (first member of theanalyte binding pair). Representative analyte binding agents includeantibodies (or fragments thereof); haptens; drug conjugates; receptors;or other binding partners. In one preferred embodiment, the analytebinding agent is an antibody to the analyte of interest. Antibodies canbe monoclonal antibodies or polyclonal antibodies. The term “antibody”,as used herein, also refers to antibody fragments which are sufficientto bind to the analyte of interest. Alternatively, in anotherembodiment, molecules which specifically bind to the analyte ofinterest, such as engineered proteins having analyte binding sites, canalso be used (Holliger, P. and H. R. Hoogenbloom, Trends inBiotechnology 13:7-9 (1995); Chamow, S. M. and A. Ashkenazi, Trends inBiotechnology 14:52-60:1996)). In still another embodiment, if theanalyte of interest is a drug, a hapten or other drug conjugate can beused as the analyte binding agent. Alternatively, in a furtherembodiment, a receptor which binds to the analyte can be used (e.g., ifthe analyte of interest is a ligand). If the analyte is an antibody ofknown specificity, the particles can be coated with the antigen againstwhich the analyte-antibody is directed, or can be coated with antibodyto the analyte-antibody. Furthermore, because the analyte and theanalyte binding agent form a binding pair, compounds or moleculesdescribed as representative analytes can also serve as analyte bindingagents, and those described as representative analyte binding agents cansimilarly serve as analytes, as described herein.

In one embodiment, the particles are also coated with a calibrationanalyte binding agent that is a second member of the calibration analytebinding pair. As described above, the calibration analyte binding agent(second member of the calibration analyte binding pair) specifically andpreferentially binds to the calibration analyte (first member of thecalibration analyte binding pair). Representative calibration analytebinding agents include those types of binding agents described abovewith regard to analyte binding agents, including antibodies (orfragments thereof); haptens; drug conjugates; receptors; or otherbinding partners. In one preferred embodiment, the calibration analytebinding agent is an antibody to the calibration analyte. Antibodies canbe monoclonal antibodies or polyclonal antibodies, as described above.In still another embodiment, if the calibration analyte is a drug, ahapten or other drug conjugate can be used as the calibration analytebinding agent. Alternatively, in a further embodiment, a receptor whichbinds to the calibration analyte can be used (e.g., if the calibrationanalyte is a ligand). If the calibration analyte is an antibody of knownspecificity, the particles can be coated with the antigen against whichthe analyte-antibody is directed, or can be coated with antibody to theanalyte-antibody. Furthermore, because the calibration analyte and thecalibration analyte binding agent form a binding pair, compounds ormolecules described as representative calibration analytes can alsoserve as calibration analyte binding agents, and those described asrepresentative calibration analyte binding agents can similarly serve ascalibration analytes, as described herein.

If the particles are coated both with analyte binding agent andcalibration analyte binding agent, they can be prepared by mixing theanalyte binding agent and calibration analyte binding agent in aconjugation buffer, so that a homogeneous solution is made. A covalentcoupling onto the particles is then performed, resulting in randombinding of the binding agents onto the particle, in proportion to theamounts in solution.

In a second embodiment, the sample collection apparatus contains apopulation of analyte binding particles coated with an analyte bindingagent, as described above, and also contains a population of calibrationanalyte binding particles that are coated with a calibration analytebinding agent that is a second member of the calibration analyte bindingpair. As described above, the calibration analyte binding agent (secondmember of the calibration analyte binding pair) specifically andpreferentially binds to the calibration analyte (first member of thecalibration analyte binding pair). Representative calibration analytebinding agents include those types of binding agents described abovewith regard to analyte binding agents, including antibodies (orfragments thereof); haptens; drug conjugates; receptors; or otherbinding partners. In one preferred embodiment, the calibration analytebinding agent is an antibody to the calibration analyte. Antibodies canbe monoclonal antibodies or polyclonal antibodies, as described above.In still another embodiment, if the calibration analyte is a drug, ahapten or other drug conjugate can be used as the calibration analytebinding agent. Alternatively, in a further embodiment, a receptor whichbinds to the calibration analyte can be used (e.g., if the calibrationanalyte is a ligand). If the calibration analyte is an antibody of knownspecificity, the particles can be coated with the antigen against whichthe analyte-antibody is directed, or can be coated with antibody to theanalyte-antibody. Furthermore, because the calibration analyte and thecalibration analyte binding agent form a binding pair, compounds ormolecules described as representative calibration analytes can alsoserve as calibration analyte binding agents, and those described asrepresentative calibration analyte binding agents can similarly serve ascalibration analytes, as described herein.

The binding particles (coated with both analyte binding agent andcalibration analyte binding agent), or the analyte binding particles andthe calibration analyte binding particles, are stored within the samplecollection apparatus in a stable form. A “stable form,” as the term isused herein, indicates a form in which the particles do notsignificantly change in chemical makeup or physical state duringstorage. The stable form can be a liquid, gel, or solid form. Inpreferred embodiments, the particles are evaporatively dried;freeze-dried; and/or vacuum-dried. The analyte binding particles and thecalibration analyte binding particles are stored at the same locationwithin the sample collection apparatus (e.g., applied as a homogeneousmixture to the location).

In one further embodiment of the invention, calibration antigen (asdescribed above) is also stored within the sample collection apparatusin a stable form. If the sample collection apparatus contains thecalibration antigen, then the calibration antigen is stored at aseparate location in the sample collection apparatus from the bindingparticles or from the calibration antigen binding particles. If thecalibration antigen is not stored within the sample collectionapparatus, then it is present in a buffer used in the methods of theinvention, as described below.

In a particularly preferred embodiment, the sample collection apparatusis a pipette tip in which the particles (and the calibration antigen, ifapplicable) are vacuum-dried or lyophilized.

To perform the assays, a sample to be assessed for the presence of theanalyte of interest, as described above, is used. In one embodiment, thesample is introduced into (drawn into, poured into, or otherwise placedinto) the sample collection apparatus. For example, in one embodiment, afluid sample is drawn up into a sample collection apparatus thatcomprises a pipette tip. In the embodiment in which binding particles(coated with analyte binding agent and calibration analyte bindingagent) are present in the collection apparatus, introduction of a fluidsample into the sample collection apparatus results in mixing of thefluid sample with the binding particles. If calibration analyte is alsopresent in the collection apparatus, introduction of a fluid sample intothe sample collection apparatus similarly results in mixing of the fluidwith the calibration analyte (in addition to the binding particles). Themixing results in the formation of a mixed fluid sample comprisingcalibration analyte. If the binding particles (and the calibrationanalyte, if present) are evaporatively-, freeze- or vacuum-dried, theintroduction of the fluid sample into the sample collection apparatuscan result in rehydration and suspension of the binding particles (andthe calibration analyte, if present) in the fluid sample.

In the embodiment in which the sample collection apparatus contains bothanalyte binding particles and calibration analyte binding particles,introduction of a fluid sample into the sample collection apparatusresults in mixing of the fluid sample with both types of bindingparticles. As before, if calibration analyte is also present in thecollection apparatus, introduction of the fluid sample into the samplecollection apparatus similarly results in mixing of the fluid with thecalibration analyte (in addition to the binding particles) and formationof a mixed fluid sample. If the analyte binding particles and/or thecalibration analyte binding particles are evaporatively-, freeze- orvacuum-dried, the introduction of the fluid sample into the samplecollection apparatus can result in rehydration and suspension of theparticles (and the calibration analyte, if present) in the fluid sample.

A buffer (e.g., for dilution) is also introduced, forming a “buffered,mixed fluid sample.” The buffered, mixed fluid sample can be formedeither by dispensing the mixed fluid sample into a “buffer container”(e.g., test tube) containing the buffer, or by introducing the bufferinto the sample collection apparatus prior to introducing a fluidsample. Alternatively, if desired, the buffer can be first introducedinto the sample collection apparatus, followed by introduction of afluid sample into the sample collection apparatus.

If the analyte of interest is a solid (e.g., a powder, a particulate;spore; or other particle, as described above), a fluid sample asdescribed above can be prepared by introducing the solid into the buffercontainer; in this embodiment, the buffered, mixed fluid sample isformed by introducing the fluid sample (comprising the buffer) into thesample collection apparatus. In another embodiment, the buffer isintroduced into the sample collection apparatus, followed byintroduction of the sample into the sample collection apparatus.

The buffer can be an aqueous fluid that supports a reaction between theanalyte of interest and the analyte binding agent (e.g., does notinterfere with antibody/antigen interaction); and that has a viscositythat is sufficiently low to allow movement of the fluid by capillaryaction. In one embodiment, the buffer contains one or more of thefollowing components: a buffering agent (e.g., phosphate); a salt (e.g.,NaCl); a protein stabilizer (e.g., BSA, casein, serum); and/or adetergent such as a nonionic detergent or a surfactant (e.g., one ormore of the following agents commonly available in surfactant tool kits:NINATE 411, Zonyl FSN 100, Aerosol OT 100%, GEROPON T-77, BIO-TERGEAS-40, STANDAPOL ES-1, Tetronic 1307, Surfnyol 465, Surfynol 485,Surfynol 104PG-50, IGEPAL CA210, TRITON X-45, TRITON X-100, TRITON X305,SILWET L7600, RHODASURF ON-870, Cremophor EL, TWEEN 20, TWEEN 80, BRIJ35, CHEMAL LA-9, Pluronic L64, SURFACTANT 10G, SPAN 60, CREL).Optionally, if desired, the buffer can contain a thickening agent. Suchcomponents for buffers are commercially available. Representativebuffers include, for example, saline, or 50 mM Tris-HCl, pH 7.2.Alternatively, water can be used in lieu of a buffered solution; as usedherein, the term “buffer” refers to either a buffered solution or towater. If calibration analyte was not present in the sample collectionapparatus, then calibration analyte is present in the buffer in a knownamount. In certain embodiments, the calibration analyte can be presentin the buffer in an amount such as 10-100 ng/ml.

To disperse the binding particles or the analyte binding particles andcalibration analyte binding particles further into the fluid sample, ifdesired, the sample collection apparatus into which the fluid sample andthe buffer has been introduced, or the buffer container into which themixed fluid sample has been introduced, can be agitated (e.g., vortexed,shaken, pipetted down and up, etc.).

In one preferred embodiment, the sample collection apparatus comprises apipette tip having vacuum-dried binding particles within its tip; thefluid sample is drawn into the pipette, thereby rehydrating the driedbinding particles and forming a mixed fluid sample. In a particularlypreferred embodiment, the mixed fluid sample is introduced into a buffercontainer, resulting in a buffered mixed fluid sample; the bufferedmixed fluid sample in the buffer container is pipetted up and down usingthe sample collection apparatus, thereby further dispersing the analytebinding particles. In another preferred embodiment, the samplecollection apparatus comprises a pipette tip having vacuum-dried analytebinding particles and vacuum-dried calibration analyte binding particleswithin its tip; the fluid sample is drawn into the pipette, therebyrehydrating both types of dried particles and forming a mixed fluidsample. In a particularly preferred embodiment, the mixed fluid sampleis introduced into a buffer container, resulting in a buffered mixedfluid sample; the buffered mixed fluid sample in the buffer container ispipetted up and down using the sample collection apparatus, therebyfurther dispersing the analyte binding particles and calibration analytebinding particles.

The buffered, mixed fluid sample contains “contacted binding particles.”The particles (either binding particles, or analyte binding particlesand calibration analyte binding particles) are “contacted” in that theyhave been exposed to the analyte of interest (if it is present in thesample) and the calibration analyte. Contact allows binding to occurbetween the analyte of interest and the analyte binding agent (either onthe binding particles or on the analyte binding particles), and alsoallows binding to occur between the calibration analyte and thecalibration analyte binding agent (either on the binding particles or onthe calibration analyte binding particles). “Binding” of analyte or ofcalibration analyte indicates that the analyte or calibration analyteinteracts with (binds to) the analyte binding agent or to thecalibration analyte binding agent, respectively. Contacted bindingparticles may or may not have analyte of interest bound to the analytebinding agent; similarly, contacted binding particles may or may nothave calibration analyte bound to the calibration analyte binding agent.

Because there are multiple binding sites for calibration analyte on thebinding particles or on the calibration analyte binding particles, thepresence and the concentration of calibration analyte bound to eachparticle can vary; the concentration of calibration analyte bound to theparticles increases proportionally with the amount of calibrationanalyte present in the fluid sample, and the probability of a particlebeing arrested in the calibration capture zone (as described below)similarly increases with increasing amount of calibration analyte boundto the calibration analyte binding agent on the particles. Thus, thepopulation of contacted calibration analyte binding particles maycomprise particles having various amount of calibration analyte bound tothe calibration analyte binding agent, as well as particles having nocalibration analyte bound to the calibration analyte binding agent (justas the particles initially have no calibration analyte bound to thecalibration analyte binding agent). Furthermore, the degree of bindingincreases as the time factor of the conditions increases: while themajority of binding occurs within one minute (e.g., 60 seconds,preferably less than 60 seconds (e.g., 45 seconds, 30 seconds, or less),additional incubation (e.g., more than one minute (2 minutes, 5 minutes,10 minutes, 15 minutes) results in additional binding. In preferredembodiments, the solution is mixed thoroughly to allow resuspension ofparticles and homogenization of the sample (e.g., in approximately 1-2minutes). It is noted that the time kinetics are similar for the antigenbinding agent and for the calibration analyte binding agent, so althoughbinding will increase over time, the ratio (as discussed below) willremain the same.

If analyte of interest is present in the buffered, mixed fluid sample,binding occurs between the analyte and the analyte binding agent (eitheron the binding particles or on the analyte binding particles). Contactedbinding particles (or contacted analyte binding particles) may or maynot have analyte bound to the analyte binding agent, depending onwhether or not analyte is present in the fluid sample and whetheranalyte has bound to the analyte binding agent on the binding particlesor analyte binding particles. Because there are multiple binding sitesfor analyte on the binding particles or on the analyte bindingparticles, the presence and the concentration of analyte bound toparticles varies; the concentration of analyte bound to the particlesincreases proportionally with the amount of analyte present in the fluidsample, and the probability of a particle being arrested in the samplecapture zone (as described below) similarly increases with increasingamount of analyte bound to the analyte binding agent on the particles.Thus, the population of contacted binding particles may compriseparticles having various amount of analyte bound to the analyte bindingagent, as well as particles having no analyte bound to the analytebinding agent (just as the particles initially have no analyte bound tothe analyte binding agent). Furthermore, the degree of binding increasesas the time factor of the conditions increases: while the majority ofbinding occurs within one minute (e.g., 60 seconds, preferably less than60 seconds (e.g., 45 seconds, 30 seconds, or less), additionalincubation (e.g., more than one minute (2 minutes, 5 minutes, 10minutes, 15 minutes) results in additional binding.

The buffered, mixed fluid sample is applied to the application point ofthe membrane strip of the solid phase apparatus, or to the applicationpad, if present. After the membrane strip is contacted with thebuffered, mixed fluid sample, the membrane strip is maintained underconditions which allow fluid to move by capillary action to and throughthe membrane. The particles (and analyte, if present in the sample) movethrough the membrane as a result of capillary action of the fluid fromthe buffered, mixed fluid sample, to and through the “sample capturezone” on the membrane and subsequently to and through the “calibrationcapture zone.” The membrane strip is maintained under conditions (e.g.,sufficient time and fluid volume) which allow the particles to move bycapillary action along the membrane to and through the sample capturezone and subsequently to the calibration capture zone, and subsequentlybeyond the capture zones (e.g., into a wicking pad), thereby removingany non-bound particles from the capture zones.

The movement of some of the binding particles (or analyte bindingparticles) is arrested by interaction of the sample capture reagent inthe sample capture zone, with analyte of interest that is bound to theparticles, resulting in immobilization of particles in the samplecapture zone; the movement of some of the binding particles orcalibration analyte binding particles is arrested by interaction of thecalibration capture reagent in the calibration capture zone, withcalibration analyte that is bound to the particles, resulting inimmobilization of particles in the calibration capture zone. Capillaryaction subsequently moves any particles that have not been arrested ineither the sample capture zone or the calibration capture zone, onwardsbeyond the calibration capture zone. In a preferred embodiment, thefluid moves any particles that have not been arrested in either capturezone into a wicking pad which follows the calibration capture zone.

If desired, a secondary wash step can be used. A buffer (e.g., thebuffer described above) can be applied at the application point afterthe buffered, mixed fluid sample has soaked in to the membrane or intothe application pad, if present. The secondary wash step can be used atany time thereafter, provided that it does not dilute the buffered,mixed fluid sample. A secondary wash step can contribute to reduction ofbackground signal when the binding particles are detected, as describedbelow.

The amount of particles arrested in the sample capture and thecalibration capture zone are then detected. The particles are detectedusing an appropriate means for the type of label used on the particles.In a preferred embodiment, the amount of particles is detected by anoptical method, such as by measuring the amount of fluorescence of thelabel of the particles. The amount of particles arrested in thecalibration capture zone is detected in the same manner as the amount ofparticles in the sample capture zone. In a preferred embodiment, theamount is detected by an optical method, such as by measuring the amountof fluorescence of the label of the particles. Alternatively, the amountof particles can be detected using electrical conductivity or dielectric(capacitance). Alternatively, electrochemical detection of releasedelectroactive agents, such as indium, bismuth, gallium or telluriumions, as described by Hayes et al. (Analytical Chem. 66:1860-1865(1994)) or ferrocyanide as suggested by Roberts and Durst (AnalyticalChem. 67:482-491 (1995)) can be used. For example, if liposomes areused, ferrocyanide encapsulated within the liposome can be released byaddition of a drop of detergent at the capture zone, and the releasedferrocyanide detected electrochemically (Roberts and Durst, id.). Ifchelating agent-protein conjugates are used to chelate metal ions,addition of a drop of acid at the capture zone will release the ions andallow quantitation by anodic stripping voltametry (Hayes et al., id.).

In one embodiment, as described above, the amount of particles isrepresented by a curve that is directly related to the amount of labelpresent at positions along the solid phase (e.g., the membrane strip).For example, the amount of particles at each position on the membranestrip (e.g., at the sample capture zone and the calibration capturezone, and/or areas in between or adjacent to the sample capture zone andthe calibration capture zone, and/or other areas of the membrane strip)can be determined and plotted as a function of the distance of theposition along the membrane strip. The amount of particles can then becalculated as a function of the area under the curve, which is relatedto the amount of label present.

A corrected particle amount is determined, and the amount of analyte canthen be determined from the corrected particle amount using appropriatecalculation. The corrected particle amount is based on the amount ofparticles arrested in the sample capture zone and in the calibrationcapture zone. For example, in one embodiment, the corrected particleamount is inversely proportional to a ratio (R) of the particle amountpresent in the sample capture zone to the particle amount present in thecalibration capture zone. The amount of analyte present can be thendetermined from the corrected particle amount (the ratio), utilizing astandard curve. The standard curve is generated by preparing a series ofcalibration samples, containing known concentrations of the analyte ofinterest in the fluid in which the analyte is to be detected (such asserum depleted of the analyte). The assay can then performed on theseries of calibration samples; the value of R is measured for eachcalibration sample; and the R values are plotted as a function of theconcentration of analyte included in the calibration sample. Samplescontaining an unknown amount of analyte (the “test samples”) are assayedby measuring the value of R for the test sample, and the concentrationof analyte in the test sample is determined by referring to the standardcurve. As above, one standard curve can be generated and used for alltest samples in a lot (e.g., for all test samples using a specifiedpreparation of test reagents); it is not necessary that the standardcurve be re-generated for each test sample. In another embodiment, thecorrected particle amount is inversely proportional to a ratio (R) ofthe amount of the particle amount present in the sample capture zone, tothe sum of the particle amount present in the calibration capture zoneand the particle amount present in the sample capture zone. The amountof analyte present can be then determined from corrected particle amount(the ratio), utilizing a standard curve. Alternatively, other ratiosand/or standard curves can also be used to determine the amount ofanalyte in the sample. In addition, if desired, the amount of label thatis present in the background can be subtracted from the analyte coatedparticle amount present in the sample capture zone and the analytecoated particle amount present in the calibration capture zone prior tocalculation of the ratio (R).

Benefits of the Invention

The methods of the invention provide assays with enhanced sensitivity,when compared with assays in which the particles and the calibrationagents are imbedded within the membrane of the solid phase apparatus.For the assays, because the fluid sample to be assayed for the analyteof interest is mixed with the binding particles or analyte bindingparticles prior to application to the membrane, there is a longer timefor the analyte of interest to bind to the binding particles or to theanalyte binding particles prior to the capture reaction which occurs inthe membrane. Furthermore, because the interaction between the analyteof interest and the binding particles or analyte binding particlesoccurs in the fluid phase, it allows more efficient binding because ofgreater mobility of the particles, than the same interaction betweenanalyte of interest and binding particles or analyte binding particleswould be in the matrix of the membrane of the solid phase apparatus. Inaddition, the methods correct for effects of various environmentalconditions, including temperature effects, humidity effects, and otherconditions.

Also, a greater number of particles can be included in a fluidcollection apparatus than would be possible to embed in a solid phaseapparatus; the greater number further enhances the sensitivity of thereaction. In addition, because the analyte binding particles (or bindingparticles) are dispersed in the buffered, mixed fluid sample prior toapplication of the buffered, mixed fluid sample to the solid phasemembrane, the particles pass over the capture zone(s) in a continuousmanner through the capillary action of the fluid, rather than in a quickwave on the crest of a fluid front. As a result, a lower concentrationof particles flows through the capture zone(s) for a longer time: thusthe time during which particles can be “captured” is effectivelyincreased, while the amount of particles that pass through the capturezone(s) is effectively lowered, thereby avoiding the blocking of captureof some particles by others which occurs when the particles pass on thecrest of a fluid front.

In addition, the calibration reaction closely mimics the analyte ofinterest reaction, in that both reactions require two separate bindingevents: binding of analyte to analyte binding agent; and subsequentbinding of analyte to capture reagent. The kinetics of the two bindingevents are extremely similar, and allow more accurate correction ofbinding particle amounts than would occur with a one-step calibrationreaction.

Although the assays of the invention have been described particularly inrelation to immunoassays, the assays can similarly be used with otherbinding pairs as described above (e.g., nucleic acids, receptor-ligands,lectin-sugars), using the same methods as described above with thedesired components as the analyte and the analyte binding agent.

Kits of the Invention

The invention also includes kits for use in the methods describedherein. Kit components can include: first and/or second members of aspecific binding pair, buffers and/or buffer containers, fluidcollection means, one or more solid phase apparatus (optionallycomprising an application pad and/or wicking pad), at least one samplecollection apparatus, one or more buffer containers, calibration samplesfor generation of a standard curve and/or other standard curveinformation, binding particles, analyte binding particles, calibrationanalyte binding particles, capture reagents, antibodies, tools to assistin collecting of samples to be assessed for analyte of interest (e.g.,swabs), disposal apparatus (e.g., biohazard waste bags), and/or otherinformation or instructions regarding the sample collection apparatus(e.g., lot information, expiration date, etc.).

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLE 1

Assays were performed to assess a two-step internal calibration reactionwhich was designed to mimic the reaction of the analyte of interestwithin a single test strip. The immunoassays were based on RAMP™ assays(Response Biomedical). Immunoassay strips were prepared by stripingantibodies specific for the target antigen (Goat anti-CP4 polyclonalantibody J7413 provided by Monsanto Company) at the sample capture zoneat 1 mg/ml, 1 ul/cm; and antibodies specific for the calibration analyte(Rabbit anti-OA polyclonal antibody provided by UK-MOD) at thecalibration binding zone at 1 mg/ml, 1 ul/cm on Millipore HF-75nitrocellulose membrane. The membrane was blocked using 0.1% PVA 9000 in10 mM PB, pH5.5. After drying, the strips were cut and assembled insidea cartridge and a sample pad and wicking pad was placed on opposite endsof the strip. The same antibodies, as above, for the target antigen andthe calibrator were covalently conjugated to fluorescently tagged latexbeads at a concentration of 0.5 mg of antibody per 4 ml of latex. Thelatex conjugates were vacuum dried into pipette tips. A sample wasprepared consisting of both the antigen of interest and the calibrationanalyte at 10 ng/ml in buffer consisting of PBS, BSA, and Cremophol ELdetergent. The latex conjugates were introduced into the sample byreconstituting the dried latex in the sample buffer and the sample wasapplied onto the sample pad of the cartridges containing the assaystrips. The cartridges were scanned on a reader after 14 minutes. Wetested the assay at room temperature and 37° C. to determine the abilityof the modified system to correct for temperature effects.

Results are shown in FIG. 1-6. TL=Test Line (sample capture zone);ISL=Internal Standard Line (calibration capture zone); R10=Ratio(TL/TL+ISL). FIG. 2 depicts the effect of temperature on capture ofparticles at the sample capture zone; FIG. 3 depicts the effect oftemperature on capture of particles at the calibration capture zone.FIG. 1 depicts ratios that indicate the occurrence of compensation fortemperature effects in the assays. FIG. 5 depicts the effect oftemperature on capture of particles at the sample capture zone for avariety of concentrations of analyte; FIG. 6 depicts the effect oftemperature on capture of particles at the calibration capture zone fora variety of concentrations of analyte; and FIG. 4 depicts ratios thatindicate the occurrence of compensation for temperature effects in theassays.

EXAMPLE 2

In order to assess further the internal controls, a RAMP® BNP assayutilizing an anti-KLH calibration capture zone was assessed.

Overview of the RAMP® BNP Assay

The RAMP® BNP (B-type Natriuretic Protein) Assay is a quantitativeimmunochromatographic test for the determination of BNP levels in EDTAwhole blood. Diluted EDTA whole blood was added to the sample well of atest cartridge which housed the immunochromatographic test strip(membrane strip). The red blood cells were retained in a sample pad,while the separated plasma migrated along the strip. Fluorescent-dyedlatex particles coated with anti-BNP antibodies (binding particles)would bind to BNP, if present in the sample. As the sample migratedalong the strip, BNP bound particles were immobilized at the samplecapture zone, and additional particles were immobilized at thecalibration capture zone. The RAMP Reader (Response Biomedical) thenmeasured the amount of fluorescence emitted by the contacted bindingparticles bound at the sample capture zone and at the calibrationcapture zone. Using a ratio between the two fluorescence values, aquantitative reading was calculated and displayed by the reader.

The anti-KLH Calibration Capture Zone and the RAMP BNP Assay

Anti-human BNP monoclonal antibody obtained from Shionogi & Co., Ltd andKLH (Hemocyanin from Keyhole Limpets) obtained from Sigma-Aldrich Co.were covalently coupled to fluorescent-dyed latex particles at a ratioof 5 parts to 1 part. The resulting conjugate was diluted, then spottedand dried in assay specific reagent tips (RAMP BNP Assay Tip).

A second anti-human BNP monoclonal antibody obtained from Shionogi &Co., Ltd was striped onto mylar backed nitrocellulose membrane at thesample capture zone. Six mm above the sample capture zone, the sameantibody amount of anti-KLH obtained from Sigma-Aldrich Co. was stripedat the calibration capture zone. The nitrocellulose was then processed,cut and assembled into single-use, disposable plastic housings (RAMP BNPTest Cartridge) along with a sample pad at the distal end and anabsorbent sink pad at the terminal end.

Using a supplied fixed volume pipette, the RAMP BNP Assay Tip was usedto transfer an aliquot of the EDTA blood sample to a single-use SampleBuffer Vial. During a short mixing process, the assay specific reagentswere released. Using the same pipette and tip, an aliquot of theprepared sample was introduced to the sample well of the RAMP BNP TestCartridge.

Sample was metered onto the assay strip by a sample pad at the bottom ofthe sample well. As the sample was drawn by capillary action along themembrane, any target analyte (BNP) in the sample interacted and boundwith the fluorescently dyed latex particles coated with analyte specificantibody and the KLH antigen (the binding particles), formingantibody/antigen complexes (e.g., one type of contacted bindingparticles). These labeled complexes were subsequently captured at thesample capture zone when BNP was present in the sample. Complexes notbound at the detection zone would continue to travel towards theterminal end of the assay strip where they would be captured by theanti-KLH calibration capture zone recognizing the KLH antigen also boundto the latex particles.

The absorbent pad at the terminal end of the assay strip soaked upexcess sample. A section of the bottom of the Test Cartridge was open sothat the test strip could be scanned by the fluorescence scanningReader. After a fixed incubation period controlled by the Reader, theReader scanned the test strip and measured fluorescence in the samplecapture zone and the calibration capture zone. The absoluteconcentration of the BNP in the sample was determined by calculating aratio between the two zones and referring it to a lot specific standardcurve that resided in the Reader memory. The Reader integrated the areaof fluorescence under each of the scan areas. The test result was thendisplayed on the RAMP Reader LCD.

RAMP BNP Assay with anti-KLH Calibration Capture Zone Example Data

FIG. 7 depicts a typical scan profile from the RAMP BNP Assay. After afixed incubation period the Reader integrates the area of fluorescenceunder each of the scan areas.

Table 1 shows the integrated sample capture zone calibration capturezone values from which the RAMP Ratio was calculated for a typical RAMPBNP standard curve run at replicates of five at concentrations of 0, 4,10, 40, 150, 600 and 2000 pg/mL BNP. A mathematical algorithm was thenapplied to the Ratio data to solve for BNP predicted values in units ofpg/mL. It can be noted that as the concentration of BNP in the sampleincreased, so did the amount of

binding at the sample capture zone, while the amount of binding at thecalibration capture zone decreased. The calculated Ratio also increased.TABLE 1 RAMP BNP Assay, Standard Curve Data Sample Sample CalibrationPrediction (BNP Capture Capture (BNP Scan # pg/mL) Zone Zone Ratiopg/mL) 1 0 23 2778 492 0.0 2 0 23 3314 413 0.0 3 0 19 2737 413 0.0 4 0 92853 188 0.0 5 0 22 2905 450 0.0 6 4 39 2635 875 3.7 7 4 40 2978 795 3.08 4 29 2277 754 2.7 9 4 37 2596 843 3.5 10 4 35 2852 727 2.4 11 10 522638 1159 6.2 12 10 58 2692 1265 7.1 13 10 63 2776 1331 7.7 14 10 612929 1224 6.8 15 10 59 2698 1284 7.3 16 40 227 2783 4524 37.4 17 40 2332840 4549 37.7 18 40 230 2799 4555 37.7 19 40 225 2749 4539 37.6 20 40240 3179 4211 34.4 21 150 797 2508 14468 155.7 22 150 834 2942 13252138.6 23 150 890 2880 14164 151.3 24 150 825 2782 13723 145.1 25 150 7482511 13771 145.8 26 600 2177 1713 33578 618.2 27 600 2059 1732 32587579.3 28 600 2351 1811 33892 631.1 29 600 2092 1751 32661 582.1 30 6002007 1497 34366 651.2 31 2000 3619 823 48883 1988.0 32 2000 3713 84648865 1984.5 33 2000 3498 729 49652 >2000 34 2000 3799 908 48425 1901.035 2000 3673 826 48984 >2000

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. A method for quantitatively measuring the amount of an analyte ofinterest in a sample, comprising: a) providing a solid phase apparatuscomprising a membrane strip comprising an application point, a samplecapture zone and a calibration capture zone, wherein the sample capturezone is between the contact region and the calibration capture zone; b)providing a sample collection apparatus that is not in fluid contactwith the solid phase apparatus, the sample collection apparatuscontaining: i) a population of binding particles, wherein the bindingparticles are coated with both an analyte binding agent and acalibration analyte binding agent, and ii) a calibration analyte,wherein the population of binding particles is located at a differentplace in the sample collection apparatus from the calibration analyte;c) either i) introducing the sample into the sample collectionapparatus, and subsequently introducing a buffer into the samplecollection apparatus; or ii) introducing a buffer into the samplecollection apparatus, and subsequently introducing the sample into thesample collection apparatus, thereby producing a buffered, mixed fluidsample containing contacted binding particles, the contacted bindingparticles comprising: binding particles having calibration analyte boundthereon and/or having analyte of interest bound thereon; and/or bindingparticles having neither calibration analyte nor analyte of interestbound thereon; d) applying the buffered, mixed fluid sample to theapplication point of the membrane strip; e) maintaining the membranestrip under conditions which allow fluid to transport contacted bindingparticles by capillary action through the strip to and through thesample capture zone, the sample capture zone having a sample capturereagent immobilized thereon, wherein the sample capture reagent can bindto analyte of interest; and allow binding of the sample capture reagentto analyte bound on the binding particles, thereby resulting inimmobilization of binding particles in the sample capture zone; f)further maintaining the membrane strip under conditions which allowfluid to transport contacted binding particles by capillary actionthrough the strip to and through the calibration capture zone, thecalibration capture zone having a calibration capture reagentimmobilized thereon, wherein the calibration capture reagent can bind tothe calibration analyte; and allow binding of the calibration capturereagent to calibration analyte bound on the binding particles, therebyresulting in immobilization of binding particles in the calibrationcapture zone; g) further maintaining the membrane strip under conditionswhich allow the fluid in the sample to transport contacted bindingparticles not immobilized in the sample capture zone or in thecalibration zone, by capillary action beyond the calibration capturezone; h) determining the amount of binding particles in the samplecapture zone and the amount of binding particles in the calibrationcapture zone; i) determining a corrected binding particle amount fromthe amount of binding particles in the sample capture zone and theamount of binding particles in the calibration capture zone, wherein theamount of analyte of interest in the sample is directly related to thecorrected binding particle amount.
 2. The method of claim 1, wherein thecorrected binding particle amount is determined as a ratio of the amountof binding particles in the sample capture zone, to the amount ofbinding particles in the calibration capture zone.
 3. The method ofclaim 1, wherein the corrected binding particle amount is determined asa ratio of the amount of binding particles in the sample capture zone,to the sum of the amount of binding particles in the calibration capturezone and the amount of binding particles in the sample capture zone.4-7. (canceled)
 8. The method of claim 1, wherein the analyte bindingagent is an antibody.
 9. (canceled)
 10. The method of claim 1, whereinthe calibration analyte binding agent is an antibody. 11-14. (canceled)15. A method for quantitatively measuring the amount of an analyte ofinterest in a sample, comprising: a) providing a solid phase apparatuscomprising a membrane strip comprising an application point, a samplecapture zone and a calibration capture zone, wherein the sample capturezone is between the contact region and the calibration capture zone; b)providing a sample collection apparatus that is not in fluid contactwith the solid phase apparatus, the sample collection apparatuscontaining a population of binding particles, wherein the bindingparticles are coated with both an analyte binding agent and acalibration binding agent; c) either i) introducing the sample into thesample collection apparatus, and subsequently introducing a buffer intothe sample collection apparatus; or ii) introducing a buffer into thesample collection apparatus, and subsequently introducing the sampleinto the sample collection apparatus, wherein the buffer contains acalibration analyte, thereby producing a buffered, mixed fluid samplecontaining contacted binding particles, the contacted binding particlescomprising: binding particles having calibration analyte bound thereonand/or having analyte of interest bound thereon; and/or bindingparticles having neither calibration analyte nor analyte of interestbound thereon; d) applying the buffered, mixed fluid sample to theapplication point of the membrane strip; e) maintaining the membranestrip under conditions which allow fluid to transport contacted bindingparticles by capillary action through the strip to and through thesample capture zone, the sample capture zone having a sample capturereagent immobilized thereon, wherein the sample capture reagent binds toanalyte of interest; and allow binding of the sample capture reagent toanalyte bound on the binding particles, thereby resulting inimmobilization of binding particles in the sample capture zone; f)further maintaining the membrane strip under conditions which allow thefluid in the sample to transport contacted binding particles bycapillary action through the strip to and through the calibrationcapture zone, the calibration capture zone having a calibration capturereagent immobilized thereon, wherein the calibration capture reagentbinds to the calibration analyte; and allow binding of the calibrationcapture reagent to calibration analyte bound on the binding particles,thereby resulting in immobilization of binding particles in thecalibration capture zone; g) further maintaining the membrane stripunder conditions which allow the fluid in the sample to transport anycontacted binding particles not immobilized in the sample capture zoneor in the calibration zone by capillary action beyond the calibrationcapture zone; h) determining the amount of binding particles in thesample capture zone and the amount of binding particles in thecalibration capture zone; i) determining a corrected binding particleamount from the amount of binding particles in the sample capture zoneand the amount of binding particles in the calibration capture zone,wherein the amount of analyte of interest in the sample is directlyrelated to the corrected binding particle amount.
 16. The method ofclaim 15, wherein the corrected binding particle amount is determined asa ratio of the amount of binding particles in the sample capture zone,to the amount of binding particles in the calibration capture zone. 17.The method of claim 15, wherein the corrected binding particle amount isdetermined as a ratio of the amount of binding particles in the samplecapture zone, to the sum of the amount of binding particles in thecalibration capture zone and the amount of binding particles in thesample capture zone. 18-21. (canceled)
 22. The method of claim 15,wherein the analyte binding agent is an antibody.
 23. (canceled)
 24. Themethod of claim 15, wherein the calibration analyte binding agent is anantibody. 25-28. (canceled)
 29. A method for quantitatively measuringthe amount of an analyte of interest in a sample, comprising: a)providing a solid phase apparatus comprising a membrane strip comprisingan application point, a sample capture zone and a calibration capturezone, wherein the sample capture zone is between the contact region andthe calibration capture zone; b) providing a sample collection apparatusnot in fluid contact with the solid phase apparatus, the samplecollection apparatus containing: i) a population of analyte bindingparticles, wherein the analyte binding particles are coated with ananalyte binding agent; ii) a population of calibration bindingparticles, wherein the calibration binding particles are coated with acalibration binding agent; and iii) a calibration analyte, wherein thepopulations of binding particles are located at a different place in thesample collection apparatus from the calibration analyte; c) either i)introducing the sample into the sample collection apparatus, andsubsequently introducing a buffer into the sample collection apparatus;or ii) introducing a buffer into the sample collection apparatus, andsubsequently introducing the sample into the sample collectionapparatus, thereby producing a buffered, mixed fluid sample containingcontacted binding particles, the contacted binding particles comprising:analyte binding particles having analyte of interest bound thereon;analyte binding particles not having analyte of interest bound thereon;calibration binding particles having calibration analyte bound thereon;and calibration binding particles not having calibration analyte boundthereon; d) applying the buffered, mixed fluid sample to the applicationpoint of the membrane strip; e) maintaining the membrane strip underconditions which allow fluid to transport contacted binding particles bycapillary action through the strip to and through the sample capturezone, the sample capture zone having a sample capture reagentimmobilized thereon, wherein the sample capture reagent binds to analyteof interest; and allow binding of the sample capture reagent to analytebound on analyte binding particles, thereby resulting in immobilizationof analyte binding particles in the sample capture zone; f) furthermaintaining the membrane strip under conditions which allow the fluid inthe sample to transport contacted binding particles by capillary actionthrough the strip to and through the calibration capture zone, thecalibration capture zone having a calibration capture reagentimmobilized thereon, wherein the calibration capture reagent binds tothe calibration analyte; and allow binding of the calibration capturereagent to calibration analyte bound on calibration binding particles,thereby resulting in immobilization of calibration binding particles inthe calibration capture zone; g) further maintaining the membrane stripunder conditions which allow the fluid in the sample to transport anycontacted binding particles not immobilized in the sample capture zoneor in the calibration zone, by capillary action beyond the calibrationcapture zone; h) determining the amount of analyte binding particles inthe sample capture zone and the amount of calibration binding particlesin the calibration capture zone; i) determining a corrected bindingparticle amount from the amount of analyte binding particles in thesample capture zone and the amount of calibration binding particles inthe calibration capture zone, wherein the amount of analyte of interestin the sample is directly related to the corrected binding particleamount.
 30. The method of claim 29, wherein the corrected bindingparticle amount is determined as a ratio of the amount of analytebinding particles in the sample capture zone, to the amount ofcalibration binding particles in the calibration capture zone.
 31. Themethod of claim 29, wherein the corrected binding particle amount isdetermined as a ratio of the amount of analyte binding particles in thesample capture zone, to the sum of the amount of calibration bindingparticles in the calibration capture zone and the amount of analytebinding particles in the sample capture zone. 32-35. (canceled)
 36. Themethod of claim 29, wherein the analyte binding agent is an antibody.37. (canceled)
 38. The method of claim 29, wherein the calibrationanalyte binding agent is an antibody. 39-42. (canceled)
 43. A method forquantitatively measuring the amount of an analyte of interest in asample, comprising: a) providing a solid phase apparatus comprising amembrane strip comprising an application point, a sample capture zoneand a calibration capture zone, wherein the sample capture zone isbetween the contact region and the calibration capture zone; b)providing a sample collection apparatus not in fluid contact with thesolid phase apparatus, the sample collection apparatus containing: i) apopulation of analyte binding particles, wherein the analyte bindingparticles are coated with an analyte binding agent, and ii) a populationof calibration binding particles, wherein the calibration bindingparticles are coated with a calibration binding agent; c) either i)introducing the sample into the sample collection apparatus, andsubsequently introducing a buffer into the sample collection apparatus;or ii) introducing a buffer into the sample collection apparatus, andsubsequently introducing the sample into the sample collectionapparatus, wherein the buffer contains a calibration analyte, therebyproducing a buffered, mixed fluid sample containing contacted bindingparticles, the contacted binding particles comprising: analyte bindingparticles having analyte of interest bound thereon; analyte bindingparticles not having analyte of interest bound thereon; calibrationbinding particles having calibration analyte bound thereon; andcalibration binding particles not having calibration analyte boundthereon; d) applying the buffered, mixed fluid sample to the applicationpoint of the membrane strip; e) maintaining the membrane strip underconditions which allow fluid to transport contacted binding particles bycapillary action through the strip to and through the sample capturezone, the sample capture zone having a sample capture reagentimmobilized thereon, wherein the sample capture reagent binds to analyteof interest; and allow binding of the sample capture reagent to analytebound on analyte binding particles, thereby resulting in immobilizationof analyte binding particles in the sample capture zone; f) furthermaintaining the membrane strip under conditions which allow the fluid inthe sample to transport contacted binding particles by capillary actionthrough the strip to and through the calibration capture zone, thecalibration capture zone having a calibration capture reagentimmobilized thereon, wherein the calibration capture reagent binds tothe calibration analyte; and allow binding of the calibration capturereagent to calibration analyte bound on calibration binding particles,thereby resulting in immobilization of calibration binding particles inthe calibration capture zone; g) further maintaining the membrane stripunder conditions which allow the fluid in the sample to transport anycontacted binding particles not immobilized in the sample capture zoneor in the calibration zone, by capillary action beyond the calibrationcapture zone; h) determining the amount of analyte binding particles inthe sample capture zone and the amount of calibration binding particlesin the calibration capture zone; i) determining a corrected bindingparticle amount from the amount of analyte binding particles in thesample capture zone and the amount of calibration binding particles inthe calibration capture zone, wherein the amount of analyte of interestin the sample is directly related to the corrected binding particleamount.
 44. The method of claim 43, wherein the corrected bindingparticle amount is determined as a ratio of the amount of analytebinding particles in the sample capture zone, to the amount ofcalibration binding particles in the calibration capture zone.
 45. Themethod of claim 43, wherein the corrected binding particle amount isdetermined as a ratio of the amount of analyte binding particles in thesample capture zone, to the sum of the amount of calibration bindingparticles in the calibration capture zone and the amount of analytebinding particles in the sample capture zone. 46-49. (canceled)
 50. Themethod of claim 43, wherein the analyte binding agent is an antibody.51. (canceled)
 52. The method of claim 43, wherein the calibrationanalyte binding agent is an antibody. 53-76. (canceled)
 77. The methodof claim 1, wherein determining a corrected binding particle amount fromthe amount of binding particles in the sample capture zone and theamount of binding particles in the calibration capture zone includessubtracting an amount of background label from the amount of bindingparticles in the sample capture zone and from the amount of bindingparticles in the calibration capture zone.
 78. The method of claim 15,wherein determining a corrected binding particle amount from the amountof binding particles in the sample capture zone and the amount ofbinding particles in the calibration capture zone includes subtractingan amount of background label from the amount of binding particles inthe sample capture zone and from the amount of binding particles in thecalibration capture zone.
 79. The method of claim 29, whereindetermining a corrected binding particle amount from the amount ofanalyte binding particles in the sample capture zone and the amount ofcalibration binding particles in the calibration capture zone includessubtracting an amount of background label from the amount of analytebinding particles in the sample capture zone and from the amount ofcalibration binding particles in the calibration capture zone.
 80. Themethod of claim 43, wherein determining a corrected binding particleamount from the amount of analyte binding particles in the samplecapture zone and the amount of calibration binding particles in thecalibration capture zone includes subtracting an amount of backgroundlabel from the amount of analyte binding particles in the sample capturezone and from the amount of calibration binding particles in thecalibration capture zone.